Lec9/10 - Enzymes Flashcards
What role can enzyme active site residues have besides catalytic activity?
Binding energy (non-covalent interactions)
In what ways can enzyme active site residues be involved in catalysis?
Proton Donors/Acceptors (charged side chains); Groups that can form covalent bonds with substrates (e.g., nucleophiles, electrophiles) and Metal (co-factor) coordination
State the Michaelis-Menten equation
V0 = Vmax[S] / Km + [S]
How is Km defined and what do lower/higher Km values indicate?
Km is a measure of the affinity of an enzyme for a particular substrate -> lower Km = tighter binding
How is Vmax defined?
Vmax is the initial velocity at which an enzyme is Saturated with substrate
What is Kcat and what does it measure?
It is the catalytic constant/turnover number (it measures the number of molecules of substrate converted to product per active site per second)
What is the specificity constant, and is lower or higher better?
Kcat / Km -> higher = “better” enzyme reaction (used to compare catalytic efficiency of different enzymes and relative abilities of different compounds to serve as a substrate for a particular enzyme)
Name the 6 Classes of Enzymes
- Oxidoreductases/Dehydrogenases
- Transferases
- Hydrolyses
- Lyases
- Isomerases
- Ligases/Synthases
What do Oxidoreductases do?
Catalyse redox reactions
What do transferases do?
Catalyse group-transfer reactions
What do hydrolyses do?
Catalyse hydrolysis (cleave a bond, with addition of water to products)
What do lyases do?
Catalyse lysis (cleavage of C-C, C-O or C-N, leaving a double bond)
What do isomerases do?
Catalyse structural change within a molecule without changing its molecular formula
What do ligases do?
Catalyse ligation of two substrates, forming a new covalent bond (requires ATP)
What feature of enzyme kinetics can suggest that an enzyme is regulated?
A sigmoidal v0 vs [S] plot (instead of hyperbolic) - this shows non-Michaelis-Menten kinetics
What is meant by an Allosteric enzyme?
They undergo conformational change, brought about by the binding of small molecules to clefts in the protein surface
What is meant by co-operativity between subunits?
The binding of a substrate to ONE active site affects SUBSEQUENT binding to all other subunits (usually more active form)
Name the two forms of an allosteric enzyme, and state how binding of certain molecules stabilises one or the other.
Binding of an activator stabilises the active form (R-form)
Binding of an inhibitor stabilises the inactive form (T-form)
Name the two molecules that regulate the PFK-1 allosteric enzyme (and what is significant about the inhibitor molecule)
ADP = activator; PEP = inhibitor (PEP is a product of a later reaction in the same pathway -> negative feedback for committed step of glycolysis)
How do ADP and PEP affect the apparent Km of PFK-1
ADP (activator) -> lower apparent Km
PEP (inhibitor) -> higher apparent Km
Define Competitive Inhibitors
Bind only to the enzyme (instead of the substrate), does not bind to ES complex
Define Uncompetitive Inhibitors
Bind exclusively to the Enzyme-Substrate Complex, reducing product formation (does not bind to enzyme)
Define Non-Competitive Inhibitors
Bind equally well to Enzyme or ES Complex, affecting the catalysis (ES -> E + P) step
Describe the effects of competitive, uncompetitive and non-competitive inhibitors on Km and Vmax
Competitive: Increase Km
Uncompetitive: Decrease Vmax and Km
Non-Competitive: Decrease Vmax
Describe the effects of competitive, uncompetitive and non-competitive inhibitors on the Lineweaver-Burk plot of 1/v0 vs 1/[s]
Competitive: steeper gradient, same y intercept (same 1/Vmax)
Uncompetitive: same gradient, greater y intercept (higher 1/Vmax)
Non-Competitive: steeper gradient, same x intercept (same 1-/Km)
What types of enzyme inhibition are common in reality?
Competitive, and Mixed (more complex, but affects Vmax and ALSO Km)
How is stereoisomerism related to enzymatic activity?
Enzymes can usually only act on one stereoisomer of a substrate
What is significant about the shape of the transition state in an enzyme-catalysed reaction?
The active site is most complementary in shape to (and binds most tightly to) the transition state -> transition state STABILISATION lowers energy barrier
How do enzymes increase the “effective” concentration of substrate?
Proximity effect: enzyme brings reacting substrates closer together
